Abstract: In this study, a fusion strain capable of simultaneously degrading herbicide atrazine and acetochlor, which can alleviate the physiological toxicity of atrazine and acetochlor to soybean seeds, was obtained by optimizing the protoplast preparation conditions of the parental strain. Firstly, the preparation technology of protoplast was optimized by single factor screening and response surface experiment. A fusion strain capable of simultaneously degrading atrazine and acetochlor was constructed by protoplast fusion method. The degradation ability of the fusion bacteria was screened and determined by high performance liquid chromatography (HPLC). The optimum growth conditions of the fusion bacteria were determined by ultraviolet spectrophotometry. The fusion bacteria were identified by 16S rDNA. The effect of the fusion strain on the simultaneous stress of atrazine and acetochlor was determined by soybean seed germination test. The results showed that strain FH-1 had the best protoplast preparation effect under the conditions of lysozyme concentration of 1.50 mg/mL, enzymolysis time of 39.15 min and enzymolysis temperature of 41.49 ℃. Strain LY-4 has the best protoplast preparation effect under the conditions of lysozyme concentration of 1.487 mg/mL, enzymolysis time of 40.70 min and enzymolysis temperature of 36.36 ℃. Using protoplast fusion technology, several fusion strains were constructed, which can degrade atrazine and acetochlor at the same time. Among them, strain RH-92 has good degradation performance, and the degradation rates of 10 mg/L acetochlor and 10 mg/L atrazine reached 61.75% and 56.18%, respectively. The phylogenetic tree identification of 16S rDNA showed that the strain RH-92 was Klebsiella quasivariicola, and the optimum growth conditions in basic salt medium were pH 9.0 and temperature 32 ℃. In the bioremediation experiment, under the simultaneous stress of atrazine and acetochlor, the germination status of soybean seeds was significantly inhibited. When RH-92 bacterial suspension was added, the germination rate and germination length of soybean increased by 25% and 25%, respectively. It shows that the fusion strain RH-92 has a good application potential in remediating the contamination caused by the coexistence of atrazine and acetochlor.